PROJECT SUMMARY During the last funding period, Project 4 (new Project 3), headed by Dr. Theresa Pizarro, in collaboration with Projects, 1, 2, and 3, has made important discoveries regarding the role of dysregulated epithelial-immune cell interactions in the pathogenesis of experimental Crohn's disease (CD). This competitive renewal represents the continuation of our efforts to understand the mechanism(s) of epithelial-immune cell interactions and the contribution of the gut microbiome during chronic intestinal inflammation and focuses on the pivotal role interleukin (IL)-33 plays in this process. The central hypothesis of Project 3 is that aberrant expression of IL-33, induced by components of the gut microbiome, mediates pathogenic Th2 immune responses through dysregulated group 2 innate lymphoid cells (ILC2s), leading to chronic CD-like ileitis. To test this hypothesis, we will: 1) Investigate the mechanism(s) as to how NOD2 activation and components of the microbiome induce IL-33-mediated Th2 immune responses leading to CD-like ileitis. We will determine the precise mechanism(s) of NOD2- dependent IL-33 regulation in SAMP by identifying which gut mucosal cells are responsible for this effect and the functional in vivo consequences of NOD2-dependent IL-33 expression. In collaboration with Project 1, using germ- free (GF)-SAMP and -AKR and FMT experiments, we will also determine which components of the gut microbiome are responsible for stimulation of intestinal mucosal IL-33 and downstream Th2 immune responses; 2) Determine the pathogenic contribution of dysregulated IL-33/ST2 signaling to the development of chronic experimental ileitis. We will investigate the precise role of IL-33 during chronic intestinal inflammation in SAMP mice using complementary ex vivo and in vivo approaches, including chromatin immunoprecipitation sequencing (ChIP-seq) and use of novel SAMP X IL-33-/- and SAMP X ST2-/- congenic mouse strains. We will determine whether nuclear localization of IL-33 is impaired in SAMP, leading to increased soluble (s)IL-33 available for proinflammatory, paracrine signaling, and if the intracrine, full length (fl)-IL-33-mediated transcriptional profile of SAMP intestinal epithelial cells (IECs) is altered in favor of proinflammatory gene targets. Finally, we will evaluate whether loss of the IL-33/ST2 signaling axis will reduce ileitis in SAMP; 3) Evaluate the role of IL-33-driven ILC2s in inducing pathogenic Th2 immune responses in SAMP ileitis. We will determine the effects of IL-33 and/or ST2 genetic deletion on ILCs in SAMP, as well as characterize ILCs in GF-SAMP and SAMP X NOD2-/- mice. We will also evaluate the functional effects of ILC2s by performing depletion studies, as well as whether reconstitution of ILC2s results in amelioration or exacerbation of ileitis and epithelial barrier function in SAMP. The overall objective of Project 3 is to further elucidate dysregulated epithelial-immune cell interactions and their interplay with the gut microbiome in the pathogenesis of SAMP ileitis and identify potential new pathways that can lead to the development of targeted strategies to treat patients suffering from this devastating disease.